LOCUS       sequence-486129-        8112 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Constitutive expression of dCas9 and alpha-NTD-MCP-SoxS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8112)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 8112)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK692
FEATURES             Location/Qualifiers
     source          1..8112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(220..298)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(594..620,621..623,624..3110,3111..3113,3114..4700)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4724..4795
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4811..4838
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4891..4925
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4935..4946
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(6458..6475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6592..7137)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6672..6691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(7251..7345)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7366..8025)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7940..7959
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(8026..8112,1..16))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 tcttacgtgc cgatcaacgt ctcattttcg ccagatatcg acgtcttaag ttacgaaatc
       61 atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga atgataaaag
      121 gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc gcttgttttt
      181 ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg gtgccacttt
      241 ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga atggttccaa
      301 caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag aaatttatca
      361 gccataaaac aatacttaat actatagaat gataacaaaa taaactactt tttaaaagaa
      421 ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa gagatatttt
      481 gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac tattattgaa
      541 atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca aaaatggata
      601 agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg
      661 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
      721 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
      781 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
      841 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
      901 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
      961 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
     1021 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
     1081 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
     1141 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
     1201 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
     1261 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1321 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     1381 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     1441 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     1501 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     1561 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     1621 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     1681 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     1741 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     1801 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     1861 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     1921 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     1981 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     2041 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     2101 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     2161 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     2221 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     2281 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     2341 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     2401 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     2461 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     2521 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     2581 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     2641 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     2701 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     2761 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     2821 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     2881 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     2941 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     3001 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     3061 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat gccattgttc
     3121 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     3181 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     3241 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     3301 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     3361 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     3421 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     3481 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     3541 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     3601 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     3661 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     3721 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa
     3781 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     3841 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag
     3901 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     3961 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     4021 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     4081 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     4141 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     4201 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     4261 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     4321 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     4381 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     4441 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     4501 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg
     4561 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt
     4621 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     4681 gtcagctagg aggtgactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc
     4741 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     4801 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctaga gcgcccggaa
     4861 gagagtcaat tcagggtggt gaatctgcag tttacggcta gctcagccct aggtattatg
     4921 ctagcgaatt cattaaagag gagaaaggta ccatggggcc catgcagggt tctgtgacag
     4981 agtttctaaa accgcgcctg gttgatatcg agcaagtgag ttcgacgcac gccaaggtga
     5041 cccttgagcc tttagagcgt ggctttggcc atactctggg taacgcactg cgccgtattc
     5101 tgctctcatc gatgccgggt tgcgcggtga ccgaggttga gattgatggt gtactacatg
     5161 agtacagcac caaagaaggc gttcaggaag atatcctgga aatcctgctc aacctgaaag
     5221 ggctggcggt gagagttcag ggcaaagatg aagttattct taccttgaat aaatctggca
     5281 ttggccctgt gactgcagcc gatatcaccc acgacggtga tgtcgaaatc gtcaagccgc
     5341 agcacgtgat ctgccacctg accgatgaga acgcgtctat tagcatgcgt atcaaagttc
     5401 agcgcggtcg tggttatgtg ccggcttcta cccgaattca ttcggaagaa gatgagcgcc
     5461 caatcggccg tctgctggtc gacgcatgct acagccctgt ggagcgtatt gcctacaatg
     5521 ttgaagcagc gcgtgtagaa cagcgtaccg acctggacaa gctggtcatc gaaatggaaa
     5581 ccaacggcac aatcgatcct gaagaggcga ttcgtcgtgc ggcaaccatt ctggctgaac
     5641 aactggaagc tttcgttgac ttacgtgatg tacgtcagcc tgaagtgaaa gaagagaaac
     5701 cagaggcttc taactttact cagttcgttc tcgtcgacaa tggcggaact ggcgacgtga
     5761 ctgtcgcccc aagcaacttc gctaacggga tcgctgaatg gatcagctct aactcgcgtt
     5821 cacaggctta caaagtaacc tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca
     5881 ccatcaaagt cgaggtgcct aaaggcgcct ggcgttcgta cttaaatatg gaactaacca
     5941 ttccaatttt cgccacgaat tccgactgcg agcttattgt taaggcaatg caaggtctcc
     6001 taaaagatgg aaacccgatt ccctcagcaa tcgcagcaaa ctccggcatc tacggtggcg
     6061 gaggtagcat gtcccatcag aaaattattc aggatcttat cgcatggatt gacgagcata
     6121 ttgaccagcc gcttaacatt gatgtagtcg caaaaaaatc aggctattca aagtggtact
     6181 tgcaacgaat gttccgcacg gtgacgcatc agacgcttgg cgattacatt cgccaacgcc
     6241 gcctgttact ggccgccgtt gagttgcgca ccaccgagcg tccgattttt gatatcgcaa
     6301 tggacctggg ttatgtctcg cagcagacct tctcccgcgt tttcgcgcgg cagtttgatc
     6361 gcactcccgc ggattatcgc caccgcctgt aagcggccgc cacgcaaaaa accccgcttc
     6421 ggcggggttt tttcgcccta gggatatatt ccgcttcctc gctcactgac tcgctacgct
     6481 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
     6541 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
     6601 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     6661 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
     6721 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     6781 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     6841 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     6901 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     6961 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     7021 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     7081 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     7141 gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat
     7201 gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg attctcacca
     7261 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
     7321 attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg
     7381 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     7441 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     7501 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     7561 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     7621 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     7681 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     7741 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     7801 catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     7861 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     7921 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     7981 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     8041 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     8101 aaagttggaa cc
//
